Stabilized coenzyme solutions and their use thereof for the determination of dehydrogenases or the substrate thereof in an alkaline medium

ABSTRACT

Stabilized aqueous coenzyme solutions which contain up to ca. 60 mM of a heavy metal salt and optionally a complexing agent in a defined ratio to the heavy metal salt as well as their use for the determination of dehydrogenases in particular lactate dehydrogenase or corresponding substrates in an alkaline environment.

The invention concerns stabilized aqueous solutions of a coenzyme forhydrogen-transferring enzymes as well as their use for determining acorresponding analyte (substrate) in a reduced form or the enzymeactivity of a corresponding dehydrogenase. The stabilized solutioncontains a heavy metal salt and optionally a completing agent as afurther component and hence enables the determination in an alkalineenvironment preferably at a pH value of 8.5 to 10.0.

The determination of enzyme activities (or substrate concentrations) inparticular in blood serum or blood plasma plays an important role inclinical chemical diagnostics. Test procedures are often used for thiswhich are based on the reduction of nicotinamide adenine dinucleotide("NAD") or nicotinamide adenine dinucleotide phosphate ("NADP") and thephotometric detection of the change of absorbance behaviour in theultraviolet wavelength range (λ=334, 340 or 365 nm) which occurs in thisprocess. When. choosing suitable test conditions this change is linearlyproportional to the enzyme activity (or substrate concentration) to bedetermined.

The method described in Eur. J. Chin. Chem. Chin. Biochem. 31, 897(1994) and Eur. J. Chin. Chem. Chin. Biochem. 32, 639 (1994) is nowadaysgenerally recommended to determine the enzyme activity of for examplelactate dehydrogenase (LDH, E.C.1.1.1.27). The test principle is thatlactate is oxidized to pyruvate with the simultaneous reduction of acoenzyme such as NAD or NADP to NADH or NADPH. Such a conversion, whichin this case is for example catalysed by LDH, takes place in an alkalinemedium (pH 9.4) i.e. under conditions under which it is known that NADor NADP is unstable. This instability is manifested by a relativelyrapid increase in absorbance (the so-called reagent blank) in therelevant wavelength range for the measurement so that the reagentcombination already becomes useless after a short period (3 months) evenwith refrigerated storage (2° to 8° C.). This is a particular problemfor the production of ready-to-use liquid reagents with long-termstability which are intended to enable the user to carry out analyses inthe daily routine as simply and reliably as possible.

A process for stabilizing aqueous coenzymes using chelating agents andazides is known from JP 84/82398. However, a disadvantage of thisprocess is that it is necessary to add azide which on the one hand hasbeen classified as cancerogenic and in addition has an inhibitory effecton many enzymes. Moreover this stabilization process has clear limits athigher pH values and temperatures.

The object of the present invention is therefore to provide an improvedstable liquid reagent containing a coenzyme for hydrogen-transferringenzymes which is suitable for the determination of dehydrogenaseactivity or of corresponding substrates.

The invention is achieved by an aqueous solution which contains a heavymetal salt at a concentration of approximately 1 to 60 mM preferably ata concentration of 2 to 10 mM. The pH value of corresponding workingsolutions is in the alkaline range preferably above pH 8.5, particularlypreferably between pH 8.5 and 10.0. Working solutions are understoodaccording to the invention to mean that the stabilized coenzyme solutionis mixed with a solution which is essentially composed of a substancebuffering in the alkaline range. It has proven to be advantageous thatthe ratio of stabilizer to buffer solution is ca. 1:5. Water-solublesalts of acids with heavy metal ions such as e.g. Cu²⁺ and/or Co²⁺ andalso other heavy metals such as for example iron, manganese, zinc,nickel, silver, magnesium, calcium or palladium have proven to besuitable as the heavy metal salt. The acid residues of common acidsknown to a person skilled in the art come into consideration as thecounterion i.e. the so-called acid anion such as for example sulfate,phosphate, the acid residues of carboxylic acids and halogenides. Copperand cobalt sulfate and/or the corresponding chlorides have proven to beparticularly suitable.

Substances which are suitable as buffers are those which have a goodbuffering capacity between ca. 8.5 and 10.0 such as for example theso-called Good buffers (tricine, bicine, TAPS, AMPSO, CHES, CAPSO, AMP,CAPS), carbonates of alkaline metal ions, MEG, TRIS as well as borate orphosphate buffer. Mixtures of the said buffer substances have alsoproven to be suitable for the solution according to the invention. Ithas proven to be suitable when the buffer concentration is between 10and 1000 mM particularly preferably between 400 and 600 mM.

The stability of the solutions can moreover be further improved when thesolution additionally contains a complexing agent i.e. a ligand whichhas two or more coordination positions. In this case bidentate ligandssuch as e.g. ethylene diamine and quadridentate and multidentate ligandssuch as ethylene diamine-N,N,N,N-tetraacetic acid (EDTA) and respectivesalts in particular the disodium salt, crown ethers or cryptands haveproven to be advantageous. It was possible to observe the stabilizingeffect especially when the complexing agent is added in a particularratio to the concentration of the heavy metal salt, the respective heavymetal salt being present in excess. The ratio of metal ion to complexingagent is preferably 2 to 1. This corresponds to a concentration of thecomplexing agent of approximately 0.5 to 30 mM preferably of 1.0 to 5.0mM.

Coenzymes which come into consideration as coenzymes within the sense ofthe present invention are in particular NAD or NADP but also modifiedcoenzymes such as for example thioNAD(P) or NH×DP (=nicotinamidehypoxanthine-dinucleotide phosphate). The coenzymes can be present at aconcentration of about 1.0 to 60 mM, a range of 5.0 to 15.0 mM ispreferred in this case.

The stabilized coenzyme solutions i.e. together with a heavy metal saltalone as well as together with a complexing agent, are preferably usedin the form of lyophilisates. However, the ready-to-use reagent is alsoadditonally stable over a long period as a granulate, powder mixture aswell as an aqueous solution. Thus at temperatures of 2° to 8° C. nomanifestations of decomposition of the reagent are found at all within15 months. Under stress i.e. at a temperature of ca. 35° C. for 2 weeksand subsequent treatment at ca. 42° C. for one day it was possible toshow that the solution containing the heavy metal salt remainedunchanged up to approximately 60 mM i.e. remained stable.

A further subject matter of the invention is a method for thedetermination of a hydrogen-transferring analyte or a correspondingdehydrogenase in the presence of a hydrogen accepting coenzyme whereinthe coenzyme is present in a stabilized aqueous alkaline solution asdescribed above. The final concentration of the heavy metal salt in thetest is in this case preferably between 0.1 and 1.0 mM; the finalconcentration of the optionally added complexing agent is preferably ca.0.05 to 5 mM.

The determination is carried out especially in samples of biologicalorigin such as for example whole blood, serum or plasma or milk or otherhuman or animal sources or in plant extracts. The sample can be preparedwith physiological saline. In such a case a 0.9% NaCl solution serves asa control value.

In the case that it is intended to determine the enzyme activity of adehydrogenase such as for example lactate dehydrogenase, a substratesolution, for example a lactate solution, is used in a substance(mixture) buffering at ca. pH 9.4 (37° C). In this case the substratecan be used in the usual concentrations known to a person skilled in theart preferably in a range of 40 to 80 mM.

In order to determine a hydrogen-transferring analyte such as forexample lactate, the respective dehydrogenase such as for example LDH isadded first in a substance buffering between pH 8.5 and 10.0. As a ruledehydrogenase in an amount of approximately 70 to 500 U/l preferably of110 to 220 U/l is adequate. The determination is usually carried out atca. 37° C.

In addition to lactate which is quoted as an example it is also possibleto determine in a similar manner glutamate or ammonia, alcohol,glyceraldehyde-3-phosphate, glucose or other parameters which can beconverted by a suitable coenzyme-dependent dehydrogenase. The sameapplies for the determination of the enzyme activity of suchdehydrogenases.

A further subject matter of the invention is a so-called test kit forcarrying out the enzyme or analyte determination. The kit is essentiallycomposed of two reagents. The first reagent comprises--in the case thatthe activity of a dehydrogenase is determined--a hydrogen-transferringanalyte (substrate) in a suitable system buffering between pH 8.5 and10.0. The second reagent contains a coenzyme for hydrogen-transferringenzymes such as for example NAD or NADP and a water-soluble heavy metalsalt at a concentration of ca. 1.0 and 60 mM. In addition the secondreagent can contain a complexing agent at a concentration of 0.5 to 30mM. An analogous second reagent should be used in the case that ananalyte or substrate is determined such as for example lactate.

Abbreviations:

    ______________________________________                                        AMP =     2-amino-2-methyl-1-propanol                                           AMPSO = 3-[(1,1-dimethyl-2-hydroxylethyl)amino-2-                              hydroxypropanesulfonic acid                                                  bicine = N,N-bis[2-hydroxyethyl]glycine                                       CAPS = 3-[(cyclohexylamino]-1-propanesulfonic acid                            CAPSO = 3-[cyclohexylamino]-2-hydroxy-1-propane-                               sulfonic acid                                                                CHES = 2-[N-cyclohexylamino]ethanesulfonic acid                               MEG = N-methylglucamine                                                       TAPS = N-Tris[hydroxymethyl]methyl-3-aminopropane-                             sulfonic acid                                                                tricine = N-Tris[hydroxymethyl]methylglycerol                                 TRIS = 2-amino-2-(hydroxymethyl)-1,3-propanol                               ______________________________________                                    

The invention is elucidated further by the following examples:

EXAMPLE 1

Reagent 1: 390 mmol/l N-methyl glucamine, pH 9.4 (37° C); 60 mmol/llithium-L-lactate.

Reagent 2: 60 mmol/l NAD(P)+5 mmol/l copper (II) sulfate as alyophilisate, powder mixture, granulate or aqueous solution.

Incubation temperature: 37±0.1° C.; measurement wavelength 340±2 nm;light path 7 mm;

Preincubation: 5 minutes; lag phase: 2 minutes; measurement period: 2minutes.

Reagent 1=250 μl; reagent 2=50 μl; sample 7 μl NaCl solution (0.9% w/v).

a) addition of 5 mmol/l copper (II) reagent 2:

    ______________________________________                                        reagent blank value in mA/min                                                                          stressed                                                (35° C. 14 days and                                                   unstressed 42° C. 1 day)                                             ______________________________________                                        NAD (aqueous sol.)                                                                            2.5      19.3                                                   NAD + 5 mmol/l Cu(II) 0.8 4.4                                                 (aqueous sol.)                                                              ______________________________________                                    

b) addition of 5 mmol/l copper (II)+EDTA to reagient 2:

    ______________________________________                                        reagent blank value in mA/min                                                                          stressed                                                (35° C. 14 days and                                                   unstressed 42° C. 1 day)                                             ______________________________________                                        NAD (aqueous sol.)                                                                            2.5      19.3                                                   NAD + 5 mmol/l Cu + 0.8 5.0                                                   2.5 mmol/l EDTA                                                               (aqueous sol.)                                                              ______________________________________                                    

EXAMPLE 2

Reagent 1 and 2 from example 1 were used with the modifiedconcentrations of copper(II) and cobalt (II) sulfate described in thefollowing.

a) addition of 0-60 mmol/l copper (II) reagent 2:

    ______________________________________                                        reagent blank value in mA/min                                                   NAD aqueous solution addition of x mmol/l Cu (II)                                                    stressed                                               unstressed (1 day 42° C.)                                            ______________________________________                                         0 mmol/l Cu (II)                                                                             1.6      5.3                                                    20 mmol/l Cu (II) -0.2   0                                                    40 mmol/l Cu (II) 0.8 1.4                                                     60 mmol/l Cu (II) 2.1 2                                                     ______________________________________                                    

b) addition of 3 mmol/1 cobalt (II) reagent 2:

    ______________________________________                                        reagent blank value in mA/min                                                                           stressed                                              unstressed (1 day 42° C.)                                            ______________________________________                                        NAD (aqueous sol.)                                                                             2.5      19.3                                                  NAD + 3 mmol/l Co (II) 0.6 18.5                                               (aqueous sol.)                                                              ______________________________________                                    

c) addition of 5 mmol/l copper (II) to reagent 2 (NADP)

1. under stress (42° C.)

    ______________________________________                                        reagent blank value in mA/min                                                                           stressed                                              unstressed (1 day 42° C.)                                            ______________________________________                                        NADP (aqueous sol.)                                                                            -0.1     1.4                                                   without Cu (II)                                                               NADP + 5 mmol/l Cu -0.8 -0.6                                                  (aqueous sol.)                                                              ______________________________________                                    

2. Stability without stress (20° C.)

    ______________________________________                                                   blank value [mA]                                                     0 months 9 months 18 months *                                               ______________________________________                                        NADP (aqueous sol.)                                                                        2.3         11.9      21.5                                         without Cu (II)                                                               NADP + 5 mmol/l 0.7 2.2 3.7                                                   Cu(II) (aqueous sol.)                                                       ______________________________________                                         * extrapolated                                                           

What is claimed is:
 1. A stabilized coenzyme solution, consistingessentially ofa heavy metal salt which is water-soluble and has aconcentration of about 1 mM to 60 mM, comprising a heavy metal cationand at least one counterion; a hydrogen accepting coenzyme having aconcentration of about 1.0 mM to about 60 mM; and a buffering solutioncomprising at least one buffer to produce a stabilized coenzyme solutionhaving a pH of at least 8.5.
 2. The coenzyme solution of claim 1,wherein the concentration of the heavy metal salt is about 2 mM to about10 mM.
 3. The coenzyme solution of claim 1, wherein the pH of thestabilized coenzyme solution is between about 8.5 and 10.0.
 4. Thecoenzyme solution of claim 1, wherein the coenzyme is selected from thegroup consisting of nicotinamide adenine dinucleotide (NAD),nicotinamide adenine dinucleotide phosphate (NADP), thioNAD, thioNADP,and nicotinamide hypoxanthine-dinucleotide phosphate.
 5. The coenzymesolution of claim 1, wherein the concentration of the coenzyme is about5.0 mM to about 15 mM.
 6. The coenzyme solution of claim 1, furthercontaining a complexing agent.
 7. The coenzyme solution of claim 6,wherein the amount of heavy metal salt in the coenzyme colution isgreater than the amount of the complexing agent in the coenzymesolution.
 8. The coenzyme solution of claim 6, wherein the ratio of theheavy metal salt to the complexing agent is about 2:1.
 9. The coenzymesolution of claim 6, wherein the concentration of the complexing agentis about 0.5 mM to about 30 mM.
 10. The coenzyme solution of claim 6,wherein the complexing agent is present in the coenzyme solution at aconcentration of about 1.0 mM to about 5.0 mM.
 11. The coenzyme solutionof claim 1, wherein the heavy metal salt is a salt of copper, cobalt,iron, manganese, zinc, nickel, silver or palladium.
 12. The coenzymesolution of claim 1, wherein the heavy metal salt is a salt of Cu²⁺ orCo²⁺.
 13. The coenzyme solution of claim 6, wherein the complexing agentis a bidentate ligand or a respective salt thereof.
 14. The coenzymesolution of claim 13, wherein the complexing agent is selected from thegroup consisting of ethylene diamine, ethylenediamine-N,N,N,N-tetraacetic acid and corresponding disodium saltthereof, crown ethers and cryptands.
 15. The coenzyme solution of claim1, wherein the ratio of the coenzyme solution to the buffering solutionis about 1:5.
 16. The coenzyme solution of claim 1, wherein the bufferis selected from the group consisting ofN-Tris[hydroxymethyl]methylglycerol, N,N-bis[2-hydroxyethyl]glycine,N-Tris[hydroxymethyl]methyl-3-aminopropane-sulfonic acid,3-[(1,1-dimethyl-2-hydroxylethyl)amino-2-hydroxypropanesulfonic acid,2-[N-cyclohexylamino]-2-hydroxy-1-propane-sulfonic acid,3-[cyclohexylamino]ethanesulfonic acid, 2-amino-2-methyl-1-propanol,3-[cyclohexylamino]-1-propanesulfonic acid, carbonates of alkaline metalions, N-methylglucamine, 2-amino-2-(hydroxymethyl)-1,3-propanol, boratebuffer, and phosphate buffer.
 17. The coenzyme solution of claim 1,wherein the concentration of buffer in the buffering solution is betweenabout 10 mM and about 1000 mM.
 18. The coenzyme solution of claim 1,wherein the concentration of buffer in the buffering solution is betweenabout 400 mM and about 600 mM.
 19. The coenzyme solution of claims 1,wherein the at least one counterion is selected from the groupconsisting of sulfate, phosphate, acid residues of carboxylic acids, andhalides.
 20. The coenzyme solution of claim 1, wherein the heavy metalsalt is selected from the group consisting of copper sulfate, cobaltsulfate, copper chloride and cobalt chloride.
 21. A lyophilizateprepared by lyophilizing the coenzyme solution of claim
 1. 22. Alyophilizate pregared by lyophilizing the coenzyme solution of claim 6.23. A ready-to-use granulate powder mixture prepared by drying thecoenzyme solution of claim
 1. 24. A ready-to-use granulate powdermixture prepared by drying the coenzyme solution of claim
 6. 25. Thecoenzyme solution of claim 1, wherein the coenzyme solution is aready-to-use aqueous solution.
 26. The coenzyme solution of claim 6,wherein the coenzyme solution is a ready-to-use aqueous solution.
 27. Amethod for determining a hydrogen-transferring analyte in a sample inthe presence of a hydrogen accepting coenzyme, comprising the stepsofmixing a buffering substance having a pH of about 8.5 to about 10.0with a dehydrogenase corresponding to the hydrogen-transferring analyteto be determined; mixing the buffering substance having thedehydrogenase therein with a stabilized coenzyme solution consistingessentially of a hydrogen accepting coenzyme and a heavy metal saltwhich is water-soluble, to obtain a mixture; and mixing thehydrogen-transferring analyte to be determined with the mixture; anddetermining the hydrogen-transferring analyte.
 28. The method of claim27, wherein the sample is a body fluid.
 29. The method of claim 28,wherein the sample is blood.
 30. The method of claim 27, wherein thehydrogen transfering analyte is lactate, the dehydrogenase is lactatedehydrogenase, and the determination is performed at a temperature ofabout 37° C.
 31. The method of claim 30, wherein about 70 U/I to about500 U/I of the lactate dehydrogenase is added to the bufferingsubstance.
 32. The method of claim 30, wherein about 110 U/I to about220 U/I of the lactate dehydrogenase is added to the bufferingsubstance.
 33. The method of claim 27, wherein the hydrogen transferinganalyte is selected from the group consisting of glutamate, ammonia,alcohol, glyceraldehyde-3-phospate, and glucose.
 34. The method of claim27, wherein the coenzyme solution further comprises a complexing agent.35. A method for determining the enzyme activity of a dehydrogenase in asample, in the presence of a hydrogen accepting coenzyme, comprising thesteps ofmixing a buffering substance having a pH of about 8.5 to about10.0 with hydrogen-transferring analyte corresponding to thedehydrogenase to be determined; mixing the buffered substance having thehydrogen-transferring analyte therein with a stabilized coenzymesolution consisting essentially of a hydrogen accepting coenzyme and aheavy metal salt which is water-soluble, to obtain a mixture; mixing thedehydrogenase to be determined with the mixture; and, determining theenzyme activity of the dehydrogenase.
 36. The method of claim 35,wherein the enzyme to be determined is lactate dehydrogenase, thehydrogen-transferring analyte is lactate, and the determination iscarried out at a pH of about 9.4 and a temperature of about 37° C. 37.The method of claim 35, wherein the coenzyme solution further comprisesa complexing agent.
 38. A test kit for the determination of a hydrogentransfering analyte in a sample, consisting essentially ofa firstreagent consisting essentially of a dehydrogenase in a buffering systemwith pH level of about 8.5 to about 10.0; and a second reagentconsisting essentially of a coenzyme for hydrogen-transferring enzymes,and a water-soluble heavy metal salt at a concentration of about 1.0 mMto about 60 mM.
 39. The test kit as in claim 38, wherein the secondreagent further comprises a complexing agent at a concentration ofbetween about 0.5 to about 30 mM.
 40. The test kit as in claim 38,wherein the first reagent and the second reagent are contained inseparate packages.
 41. A test kit for the determination of an enzymeactivity of a dehydrogenase in a sample, consisting essentially of afirst reagent consisting essentially of a hydrogen-transfering analytein a buffering system having pH level of about 8.5 to about 10.0; andasecond reagent consisting essentially of a coenzyme forhydrogen-transfering enzymes, and a water-soluble heavy metal salt at aconcentration of about 1.0 mM to about 60 mM.
 42. The test kit as inclaim 41, wherein the second reagent further comprises a complexingagent at a concentration of about 0.5 mM to about 30 mM.
 43. The testkit as in claim 41, wherein the coenzyme is selected from the groupconsisting of nicotinamide adenine dinucleotide (NAD), nicotinamideadenine dinucleotide phosphate (NADP), thioNAD, thioNADP andnicotinamide hypoxanthine-dinucleotide phosphate.
 44. The test kit as inclaim 41, wherein the first reagent and the second reagent are containedin separate packages.
 45. The method of claim 35, wherein thedehydrogenase is a dehydrogenase for a hydrogen transferring analyteselected from the group consisting of glutamate, ammonia, alcohol,glyceraldehyde-3-phosphate, and glucose.
 46. A stabilized coenzymesolution, consisting essentially ofabout 1 mM to about 60 mM of at leastone water-soluble salt of silver or palladium with at least onecounterion; and a hydrogen accepting coenzyme having a concentration ofabout 1.0 mM to about 60 mM.
 47. The coenzyme solution of claim 46,wherein the pH of the stabilized coenzyme solution is between about 8.5and about 10.0.
 48. The coenzyme solution of claim 46, wherein theconcentration of the salt of silver or palladium is about 2 mM to about10 mM.
 49. The coenzyme solution of claim 46, wherein the coenzyme isselected from NAD, NADP, thioNAD, thioNADP and nicotinamide hypoxanthinedinucleotide phosphate.
 50. The coenzyme solution of claim 46, whereinthe concentration of the coenzyme is about 5 mM to about 15 mM.
 51. Thecoenzyme solution of claim 6, wherein the complexing agent is selectedfrom the group consisting of ethylene diamine, ethylenediamine-N,N,N,N-tetraacetic acid, corresponding disodium salt thereof,crown ethers and cryptands.
 52. The method of claim 34, wherein thecomplexing agent is selected from the group consisting of ethylenediamine, ethylene diamine-N,N,N,N-tetraacetic acid, correspondingdisodium salt thereof, crown ethers and cryptands.
 53. The test kit ofclaim 42, wherein the complexing agent is selected from the groupconsisting of ethylene diamine, ethylene diamine-N,N,N,N-tetraaceticacid, corresponding disodium salt thereof, crown ethers and cryptands.54. The method of claim 37, wherein the complexing agent is selectedfrom the group consisting of ethylene diamine, ethylenediamine-N,N,N,N-tetraacetic acid, corresponding disodium salt thereof,crown ethers and cryptands.
 55. The test kit of claim 39, wherein thecomplexing agent is selected from the group consisting of ethylenediamine, ethylene diamine-N,N,N,N-tetraacetic acid, correspondingdisodium salt thereof, crown ethers and cryptands.
 56. A stabilizedcoenzyme solution, consisting ofa heavy metal salt which iswater-soluble and has a concentration of about 1 mM to 60 mM, comprisinga heavy metal cation and at least one counterion; a hydrogen acceptingcoenzyme having a concentration of about 1.0 mM to about 60 mM; and abuffering solution comprising at least one buffer to produce astabilized coenzyme solution having a pH of at least 8.5.
 57. Thecoenzyme solution of claim 56, wherein the concentration of the heavymetal salt is about 2 mM to about 10 mM.
 58. The coenzyme solution ofclaim 56, wherein the pH of the stabilized coenzyme solution is betweenabout 8.5 and 10.0.
 59. The coenzyme solution of claim 56, wherein thecoenzyme is selected from the group consisting of nicotinamide,adeninine dinucleotide (NAD), nicotinamide adenine dinucleotidephosphate (NADP), thioNAD, thioNADP, and nicotinamidehypoxanthine-dinucleotide phosphate.